BACKGROUND OF THE INVENTION
Field of the Invention
[0001] This invention relates to novel multibinding compounds (agents) that are β2 adrenergic
receptor agonists and pharmaceutical compositions comprising such compounds. Accordingly,
the multibinding compounds and pharmaceutical compositions of this invention are useful
in the treatment and prevention of respiratory diseases such as asthma and chronic
bronchitis. They are also useful in the treatment of nervous system injury and premature
labor.
References
[0002] The following publications are cited in this application as superscript numbers:
1 Hardman, J. G., et al. "The Pharmacological Basis of Therapeutics", McGraw-Hill,
New York, (1996)
2 Strosberg, A. D. "Structure, Function, and Regulation of Adrenergic Receptors" Protein Sci. 2, 1198-1209 (1993).
3 Beck-Sickinger, A. G. "Structure Characterization and Binding Sites of G-Protein-coupled
Receptors" DDT, 1, 502-513, (1996).
4 Hein, L. & Kobilka, B. K. "Adrenergic Receptor Signal Transduction and Regulation"
Neuropharmacol, 34, 357-366, (1995).
5 Strosberg, A. D. & Pietri-Rouxel, F. "Function, and Regulation of β3-Adrenoceptor"
TiPS, 17, 373-381, (1996).
6 Barnes, P. J. "Current Therapies for Asthma" CHEST, 111:17S-26S, (1997).
7 Jack, D. A. "A way of Looking at Agonism and Antagonism: Lessons from Salbutamol,
Salmeterol and other β-Adrenoceptor Agonists" Br. J. Clin. Pharmac. 31, 501-514, (1991).
8 Kissei Pharmaceutical Co. Ltd. "2-Amino-1-(4-hydroxy-2-methylphenyl)propanol derivatives"
JP-10152460 (Publication date June 9, 1998).
State of the Art
[0003] A receptor is a biological structure with one or more binding domains that reversibly
complexes with one or more ligands, where that complexation has biological consequences.
Receptors can exist entirely outside the cell (extracellular receptors), within the
cell membrane (but presenting sections of the receptor to the extracellular milieu
and cytosol), or entirely within the cell (intracellular receptors). They may also
function independently of a cell (e.g., clot formation). Receptors within the cell
membrane allow a cell to communicate with the space outside of its boundaries (i.e.,
signaling) as well as to function in the transport of molecules and ions into and
out of the cell.
[0004] A ligand is a binding partner for a specific receptor or family of receptors. A ligand
may be the endogenous ligand for the receptor or alternatively may be a synthetic
ligand for the receptor such as a drug, a drug candidate or a pharmacological tool.
[0005] The super family of seven transmembrane proteins (7-TMs), also called G-protein coupled
receptors (GPCRs), represents one of the most significant classes of membrane bound
receptors that communicate changes that occur outside of the cell's boundaries to
its interior, triggering a cellular response when appropriate. The G-proteins, when
activated, affect a wide range of downstream effector systems both positively and
negatively (e.g., ion channels, protein kinase cascades, transcription, transmigration
of adhesion proteins, and the like).
[0006] Adrenergic receptors (AR) are members of the G-protein coupled receptors that are
composed of a family of three receptor sub-types: α1
(A, B, D) α2
(A, B, C), and β
(1,2,3).
1-5 These receptors are expressed in tissues of various systems and organs of mammals
and the proportions of the α and the β receptors are tissue dependant. For example,
tissues of bronchial smooth muscle express largely β2-AR while those of cutaneous
blood vessels contain exclusively α-AR subtypes.
[0007] It has been established that the β2-AR sub-type is involved in respiratory diseases
such as such as asthma
6, chronic bronchitis, nervous system injury, and premature labor
8. Currently, a number of drugs e.g., albuterol, formoteml, isoprenolol, or salmeterol
having β2-AR agonist activities are being used to treat asthma. However, these drugs
have limited utility as they are either non-selective thereby causing adverse side
effects such as muscle tremor, tachycardia, palpitations, and restlesness
6, or have short duration of action and/or slow onset time of action.
7 Accordingly, there is a need for β2-selective AR agonists that are fast acting and
have increased potency and /or longer duration of action.
[0008] The multibinding compounds of the present invention fulfill this need.
SUMMARY OF THE INVENTION
[0009] This invention is directed to novel multibinding compounds (agents) that are agonists
or partial agonists of β2 adrenergic receptor and are therefore useful in the treatment
and prevention of respiratory diseases such as asthma and chronic bronchitis. They
are also useful in the treatment of nervous system injury and premature labor.
[0010] The compounds according to the present invention may be regarded as examples of compounds
of the formula
L
2X (I)
where one L is a ligand of general formula
and the other L is a ligand of general formula
-QAr
3
wherein the various substituents have the meanings given hereafter.
[0011] The invention provides a bivalent multibinding compound of Formula (II) or a pharmaceutically
acceptable salt thereof:
wherein:
Ar1 is a phenyl ring of formula (c):
wherein:
R4 is hydrogen, alkyl, halo, or alkoxy;
R5 is hydrogen, hydroxy, halo, or amino;
R6 is hydrogen, halo, hydroxy, alkoxy, substituted alkyl, or -NRC(O)R, wherein each
R is hydrogen or alkyl; or Ar1 is a 2,8-dihydroxyquinolin-5-yl group, which may also be represented by the formula
Ar3 is either:
(i) a phenyl ring of formula (c) as defined above; or
(ii) a phenyl ring of formula (d):
wherein:
R7 is hydrogen, alkyl, alkenyl, substituted alkyl, halo, alkoxy, substituted alkoxy,
or hydroxy; and
R8 is hydrogen, halo, alkoxy, or substituted alkoxy; or
(iii) naphthyl,
where substituted alkyl means hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-aminoethyl,
3-aminopropyl, 2-methylaminoethyl, 3-dimethylaminopropyl, 2-sulfonamidoethyl or 2-carboxyethyl,
and substituted alkoxy means (substituted alkyl)-O-;
W is a bond, or an alkylene chain wherein one or more of the carbon atoms in the
alkylene group are optionally replaced by -O-;
Ar
2 is phenylene wherein the W and the X groups are attached at the 1,2-, 1,3, and 1,4
positions of the phenyl ring; or cyclohexylene optionally substituted with methyl
and wherein the W and the X groups are attached at the 1,3, and 1,4 positions of the
cyclohexyl ring;
X is a covalent bond; and
Q is -NH-CH
2 -CH(OH)-; -NH-CH(CH
2OH)-; -(CH
2)
3-O-(CH
2)
6-NH-CH
2-CH(OH)-; or -NH-CH
2-CH(OH)-CH
2-O-.
[0012] The invention also provides a pharmaceutical composition comprising a pharmaceutically
acceptable carrier and an effective amount of a compound of formula II or a pharmaceutically
acceptable salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIGs. 1-7 illustrate synthesis of compounds of Formula (II).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0014] This invention is directed to multibinding compounds which are β2 adrenergic receptor
agonists, pharmaceutical compositions containing such compounds and methods for treating
diseases mediated by β2 adrenergic receptor in mammals. When discussing such compounds,
compositions or methods, the following terms have the following meanings unless otherwise
indicated. Any undefined terms have their art recognized meanings.
[0015] The term "alkyl" refers to a monoradical branched or unbranched saturated hydrocarbon
chain preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon
atoms, and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups
such as methyl, ethyl,
n-propyl,
iso-propyl,
n-butyl,
isobutyl,
n-hexyl,
n-decyl, tetradecyl, and the like.
[0016] The term "substituted alkyl" refers to an alkyl group as defined above, having from
1 to 5 substituents, acid preferably I to 3 substituents, selected from the group
consisting of hydroxymethyl, hydroxyethyl, hydroxypropyl, 2-aminoethyl, 3-aminopropyl,
2-methylaminoethyl, 3-dimethylaminopropyl, 2-sulfonamidoethyl, and 2-carboxyethyl.
[0017] The term "alkylene" refers to a diradical of a branched or unbranched saturated hydrocarbon
chain, preferably having from 1 to 40 carbon atoms, more preferably 1 to 10 carbon
atoms and even more preferably 1 to 6 carbon atoms. This term is exemplified by groups
such as methylene (-CH
2-), ethylene (-CH
2CH
2-), the propylene isomers (e.g., -CH
2CH
2CH
2- and -CH(CH
3)CH
2-) and the like.
[0018] The term "alkoxy" refers to the group alkyl-O-, where alkyl is as defined herein.
Preferred alkoxy groups include, by way of example, methoxy, ethoxy,
n-propoxy,
iso-propoxy,
n-butoxy,
tert-butoxy,
sec-butoxy,
n-pentoxy,
n-hexoxy, 1,2-dimethylbutoxy, and the like.
[0019] The term "substituted alkoxy" refers to the group substituted alkyl-O-, where substituted
alkyl is as defined herein.
[0020] The term "alkenyl" refers to a monoradical of a branched or unbranched unsaturated
hydrocarbon group preferably having from 2 to 40 carbon atoms, more preferably 2 to
10 carbon atoms and even more preferably 2 to 6 carbon atoms and having at least I
and preferably from 1-6 sites of vinyl unsaturation. Preferred alkenyl groups include
ethenyl (-CH=CH
2),
n-propenyl (-CH
2CH=CH
2),
iso-propenyl (-C(CH
3)=CH
2), and the like.
[0021] The term "halo" or "halogen" refers to fluoro, chloro, bromo and iodo.
[0022] The term "pharmaceutically-acceptable salt" refers to salts which retain the biological
effectiveness and properties of the multibinding compounds of this invention and which
are not biologically or otherwise undesirable. In many cases, the multibinding compounds
of this invention are capable of forming acid and/or base salts by virtue of the presence
of amino and/or carboxyl groups or groups similar thereto.
[0023] Pharmaceutically-acceptable base addition salts can be prepared from inorganic and
organic bases. Salts derived from inorganic bases, include by way of example only,
sodium, potassium, lithium, ammonium, calcium and magnesium salts. Salts derived from
organic bases include, but are not limited to, salts of primary, secondary and tertiary
amines. Also included are amines where the two or three substituents, together with
the amino nitrogen, form a heterocyclic or heteroaryl group. Examples of suitable
amines include, by way of example only, isopropylamine, trimethyl amine, diethyl amine,
tri(
iso-propyl) amine, tri(
n-propyl) amine, ethanolamine, 2-dimethylaminoethanol, tromethamine, lysine, arginine,
histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine,
N-alkylglucamines, theobromine, purines, piperazine, piperidine, morpholine, N-ethylpiperidine,
and the like. It should also be understood that other carboxylic acid derivatives
would be useful in the practice of this invention, for example, carboxylic acid amides,
including carboxamides, lower alkyl carboxamides, dialkyl carboxamides, and the like.
[0024] Pharmaceutically acceptable acid addition salts may be prepared from inorganic and
organic acids. Salts derived from inorganic acids include hydrochloric acid, hydrobromic
acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Salts derived from
organic acids include acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic
acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric
acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-toluene-sulfonic acid, salicylic acid, and the like.
[0025] The term "pharmaceutically-acceptable cation" refers to the cation of a pharmaceutically-acceptable
salt.
[0026] The term "pseudohalide" refers to functional groups which react in displacement reactions
in a manner similar to a halogen. Such functional groups include, by way of example,
mesyl, tosyl, azido and cyano groups.
[0027] The term "protecting group" or "blocking group" refers to any group which when bound
to one or more hydroxyl, thiol, amino or carboxyl groups of the compounds (including
intermediates thereof) prevents reactions from occurring at these groups and which
protecting group can be removed by conventional chemical or enzymatic steps to reestablish
the hydroxyl, thiol, amino or carboxyl group (
See., T.W. Greene and P.G.H. Wuts, "
Protective Groups in Organic Synthesis", 2
nd Ed.). The particular removable blocking group employed is not critical and preferred
removable hydroxyl blocking groups include conventional substituents such as allyl,
benzyl, acetyl, chloroacetyl, thiobenzyl, benzylidine, phenacyl, t-butyldiphenylsilyl
and any other group that can be introduced chemically onto a hydroxyl functionality
and later selectively removed either by chemical or enzymatic methods in mild conditions
compatible with the nature of the product. Preferred removable thiol blocking groups
include disulfide groups, acyl groups, benzyl groups, and the like.
[0028] Preferred removable amino blocking groups include conventional substituents such
as t-butyoxycarbonyl (t-BOC), benzyloxycarbonyl (CBZ), fluorenylmethoxy-carbonyl (FMOC),
allyloxycarbonyl (ALOC), and the like which can be removed by conventional conditions
compatible with the nature of the product.
[0029] Preferred carboxyl protecting groups include esters such as methyl, ethyl, propyl,
t-butyl etc. which can be removed by mild conditions compatible with the nature of
the product.
[0030] The term "optional" or "optionally" means that the subsequently described event,
circumstance or substituent may or may not occur, and that the description includes
instances where said event or circumstance occurs and instances where it does not.
[0031] The term "ligand" or " ligands" as used herein denotes a compound that is a binding
partner for a β2 adrenergic receptor and is bound thereto by complementarity. Preferred
ligands are those that are either β2 adrenergic receptor agonist or antagonist. The
specific region or regions of the ligand that is (are) recognized by the receptor
is designated as the "ligand domain". A ligand may be either capable of binding to
the receptor by itself, or may require the presence of one or more non-ligand components
for binding (e.g., Ca
+2, Mg
+2 or a water molecule is required for the binding of a ligand to various ligand binding
sites). Examples of ligands useful in this invention are described herein. Those skilled
in the art will appreciate that portions of the ligand structure that are not essential
for specific molecular recognition and binding activity may be varied substantially,
replaced or substituted with unrelated structures (for example, with ancillary groups
as defined below) and, in some cases, omitted entirely without affecting the binding
interaction. The primary requirement for a ligand is that it has a ligand domain as
defined above. It is understood that the term ligand is not intended to be limited
to compounds known to be useful in binding to β2 adrenergic receptor (e.g., known
drugs). Those skilled in the art will understand that the term ligand can equally
apply to a molecule that is not normally associated with β2 adrenergic receptor binding
properties. In addition, it should be noted that ligands that exhibit marginal activity
or lack useful activity as monomers can be highly active as multivalent compounds
because of the benefits conferred by multivalency.
[0032] The term "ligand" or " ligands" as used herein is intended to include the racemic
forms of the ligands as well as individual enantiomers and diasteromers and non-racemic
mixtures thereof.
[0033] The term "potency" refers to the minimum concentration at which a ligand is able
to achieve a desirable biological or therapeutic effect. The potency of a ligand is
typically proportional to its affinity for its ligand binding site. In some cases,
the potency may be non-linearly correlated with its affinity. In comparing the potency
of two drugs, e.g., a multibinding agent and the aggregate of its unlinked ligand,
the dose-response curve of each is determined under identical test conditions (e.g.,
in an
in vitro or
in vivo assay, in an appropriate animal model such a human patient). The finding that the
multibinding agent produces an equivalent biological or therapeutic effect at a lower
concentration than the aggregate unlinked ligand is indicative of enhanced potency.
[0034] The term "selectivity" or "specificity" is a measure of the binding preferences of
a ligand for different ligand binding sites (receptors). The selectivity of a ligand
with respect to its target ligand binding site relative to another ligand binding
site is given by the ratio of the respective values of K
d (i.e., the dissociation constants for each ligand-receptor complex) or, in cases
where a biological effect is observed below the K
d, the ratio of the respective EC
50's (i.e., the concentrations that produce 50% of the maximum response for the ligand
interacting with the two distinct ligand binding sites (receptors)).
[0035] The term "ligand binding site" denotes the site on the β-adrenergic receptor that
recognizes a ligand domain and provides a binding partner for the ligand. The ligand
binding site may be defined by monomeric or multimeric structures. This interaction
may be capable of producing a unique biological effect, for example, agonism, antagonism,
and modulatory effects or it may maintain an ongoing biological event, and the like.
[0036] It should be recognized that the ligand binding sites of the receptor that participate
in biological multivalent binding interactions are constrained to varying degrees
by their intra- and inter-molecular associations. For example, ligand binding sites
may be covalently joined to a single structure, noncovalently associated in a multimeric
structure, embedded in a membrane or polymeric matrix, and so on and therefore have
less translational and rotational freedom than if the same structures were present
as monomers in solution.
[0037] The terms "agonism" and "antagonism" is well known in the art. The term "modulatory
effect" refers to the ability of the ligand to change the activity of an agonist or
antagonist through binding to a ligand binding site.
[0038] The term "inert organic solvent" or "inert solvent" means a solvent which is inert
under the conditions of the reaction being described in conjunction therewith including,
by way of example only, benzene, toluene, acetonitrile, tetrahydrofuran, dimethylformamide,
chloroform, methylene chloride, diethyl ether, ethyl acetate, acetone, methylethyl
ketone, methanol, ethanol, propanol, isopropanol,
t-butanol, dioxane, pyridine, and the like. Unless specified to the contrary, the solvents
used in the reactions described herein are inert solvents.
[0039] The term "treatment" refers to any treatment of a pathologic condition in a mammal,
particularly a human, and includes:
(i) preventing the pathologic condition from occurring in a subject which may be predisposed
to the condition but has not yet been diagnosed with the condition and, accordingly,
the treatment constitutes prophylactic treatment for the disease condition;
(ii) inhibiting the pathologic condition, i.e., arresting its development;
(iii) relieving the pathologic condition, i.e., causing regression of the pathologic
condition; or
(iv) relieving the conditions mediated by the pathologic condition.
[0040] The term "pathologic condition which is modulated by treatment with a ligand" covers
all disease states (i.e., pathologic conditions) which are generally acknowledged
in the art to be usefully treated with a ligand for the β2-adrenergic receptor in
general, and those disease states which have been found to be usefully treated by
a specific multibinding compound of our invention. Such disease states include, by
way of example only, the treatment of a mammal afflicted with asthma, chronic bronchitis,
and the like.
[0041] The term "therapeutically effective amount" refers to that amount of multibinding
compound which is sufficient to effect treatment, as defined above, when administered
to a mammal in need of such treatment. The therapeutically effective amount will vary
depending upon the subject and disease condition being treated; the weight and age
of the subject, the severity of the disease condition, the manner of administration
and the like, which can readily be determined by one of ordinary skill in the art.
Representative Compounds of Formula (II):
[0042]
I. Representative bivalent multibinding compounds of Formula (II) wherein Ar
1 is 4-hydroxy-3-hydroxymethylphenyl, Ar
2 is 1,4-phenylene, X, W, Q, and Ar
3 are as defined in Table A below are:
Table A
Cpd. # |
Stereochem. at *C |
W |
X |
-Q-Ar3 (** = stereochem) |
1A |
(RS) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)phenyl ** = (S) |
2A |
(RS) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)phenyl ** = (R) |
3A |
(RS) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)phenyl ** = (RS) |
4A |
(RS) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)-(4-hydroxy-3-hydroxy-methyl)phenyl ** = (RS) |
5A |
(RS) |
-(CH2)6O- |
bond |
-(CH2)3-O -(CH2)6-NH-CH2-** CH(OH)-(4-hydroxy-3-hydroxyethyl)phenyl ** = (RS) |
6A |
(RS) |
-CH2- |
bond |
-NH-CH2-** CH(OH)-(4-hydroxy-3-hydroxy-methyl)phenyl ** = (RS) |
7A |
(R) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)phenyl ** = (S) |
8A |
(R) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)phenyl ** = (R) |
11A |
(RS) |
-(CH2)2- |
bond |
-NH-CH2-** CH(OH)-O-naphth-1-yl ** = (RS) |
III. Representative bivalent multibinding compounds of Formula (II) wherein Ar
1 is 4-hydroxy-3-hydroxy-methylphenyl, Ar
3 is (4-hydroxy-3-hydroxymethyl)phenyl, X, W, Q, and Ar
2 are as defined in Table C below are:
Table C
Cpd. # |
Stereochem. at *C |
W |
X |
Ar2 |
Q |
1C |
(RS) |
bond |
bond |
trans-1,4-cyclohexane |
-NH-CH2-** CH(OH)-** = (RS) |
V. Representative bivalent multibinding compounds of Formula (II) wherein Ar
1 is phenyl, W is -(CH
2)
2-, and Ar
2 is 1,4-phenylene and-Q-Ar
3, is [2-hydroxy-2-phenyl]ethylamino, and X is a bond are as shown in Table E below:
Table E
Cpd. # |
Stereochem. at *C |
Stereochem. at **C |
1E |
(RS) |
(RS) |
2E |
(R) |
(S) |
3E |
(R) |
(R) |
PREFERRED EMBODIMENTS
[0043] While the broadest definition of this invention is set forth in the Summary of the
Invention, certain compounds of Formula (II) are preferred.
[0044] When Ar
1 is a phenyl ring of formula (c):
R
4 is preferably hydrogen, methyl, fluoro, chloro, or methoxy;
R
5 is preferably hydrogen, hydroxy, fluoro, chloro, or amino; and
R
6 is preferably hydrogen, chloro, fluoro, hydroxy, methoxy, hydroxymethyl, or -NHCHO.
W is preferably a covalent bond, methylene, ethylene, propylene, -(CH
2)
6-O-(CH
2)
3-, or -(CH
2)
6-O; and
Ar
2 is preferably 1,4-phenylene.
Q is -NH-CH
2-*CH(OH)-; -NH-*CH(CH
2OH)-; -(CH
2)
3-O-(CH
2)
6-NH-CH
2-*CH(OH)-; or -NH-CH
2-*CH(OH)-CH
2-O- (where * is R or S stereochemistry).
[0045] Within the above preferred, more preferred group of compounds, a particularly preferred
group of compounds is that wherein:
(i) Ar3 is same as Ar1 as defined in preferred embodiments above.
Another particularly preferred group of compounds is that wherein:
(ii) Ar3 is a phenyl ring of formula (d):
wherein:
R7 is hydrogen, alkyl, alkenyl, substituted alkyl, halo, alkoxy, substituted alkoxy,
or hydroxy, preferably hydrogen, methyl, propen-2-yl, fluoro, chloro, methoxy, or
hydroxy; and
R8 is hydrogen, halo, alkoxy, or substituted alkoxy, preferably hydrogen, fluoro, chloro,
or methoxy.
(iii) Yet another particularly preferred group of compounds is that wherein Ar3 is naphthyl.
[0046] Within the above preferred, more preferred, and particularly preferred groups, even
more particularly preferred group is that wherein:
Ar1 is phenyl, 4-hydroxyphenyl, 3,4-dihydroxyphenyl, 3,4-dichlorophenyl, 2-chloro-3,4-dihydroxyphenyl,
2-fluoro-3,4-dihydroxyphenyl, 4-hydroxy-3-methoxyphenyl, 4-hydroxy-3-hydroxymethylphenyl,
4-hydroxy-3-(HCONH-)phenyl, 3-chlorophenyl, 2,5-dimethoxyphenyl, 3,5-dichloro-4-aminophenyl,
or
preferably 4-hydroxy-3-hydroxymethylphenyl, 4-hydroxy-3-(HCONH-)phenyl, 3,5-dichloro-4-aminophenyl,
or
and
Ar3 is:
preferably, phenyl or 4-hydroxy-3-hydroxymethylphenyl.
GENERAL SYNTHETIC SCHEME
[0047] Compounds of this invention can be made by the methods depicted in the reaction schemes
shown below.
[0048] The starting materials and reagents used in preparing these compounds are either
available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wisconsin,
USA), Bachem (Torrance, California, USA), Emka-Chemie, or Sigma (St. Louis, Missouri,
USA) or are prepared by methods known to those skilled in the art following procedures
set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis,
Volumes 1-15 (John Wiley and Sons, 1991); Rodd's Chemistry of Carbon Compounds, Volumes
1-5 and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes
1-40 (John Wiley and Sons, 1991), March's Advanced Organic Chemistry, (John Wiley
and Sons, 4th Edition), and Larock's Comprehensive Organic Transformations (VCH Publishers
Inc., 1989).
[0049] The starting materials and the intermediates of the reaction may be isolated and
purified if desired using conventional techniques, including but not limited to filtration,
distillation, crystallization, chromatography, and the like. Such materials may be
characterized using conventional means, including physical constants and spectral
data.
[0050] Furthermore, it will be appreciated that where typical or preferred process conditions
(i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures,
etc.) are given, other process conditions can also be used unless otherwise stated.
Optimum reaction conditions may vary with the particular reactants or solvent used,
but such conditions can be determined by one skilled in the art by routine optimization
procedures.
[0051] Additionally, as will be apparent to those skilled in the art, conventional protecting
groups may be necessary to prevent certain functional groups from undergoing undesired
reactions. The choice of a suitable protecting group for a particular functional group
as well as suitable conditions for protection and deprotection are well known in the
art. For example, numerous protecting groups, and their introduction and removal,
are described in T. W. Greene and G. M. Wuts,
Protecting Groups in Organic Synthesis, Second Edition, Wiley, New York, 1991, and references cited therein.
[0052] These schemes are merely illustrative of some methods by which the compounds of this
invention can be synthesized, and various modifications to these schemes can be made
and will be suggested to one skilled in the art having referred to this disclosure.
Preparation of a multibinding compound of Formula (I)
[0053] In general, a bivalent multibinding compound of Formula (I) can be prepared as illustrated
and described in Schemes A-D below.
[0054] A bivalent multibinding compound of Formula (I) can be prepared by covalently attaching
the ligands, L, as defined in the Summary of the Invention, as shown in Scheme A below.
[0055] In method (a), a bivalent multibinding compound of Formula (I) is prepared in one
step, by covalently attaching the ligands, L, where FG
1 and FG
2 represent a functional group such as halo, amino, hydroxy, thio, aldehyde, ketone,
carboxy, carboxy derivatives such as acid halide, ester, amido, and the like. This
method is preferred for preparing compounds of Formula (I) where the ligands are the
same.
[0056] In method (b), the compounds of Formula (I) are prepared in a stepwise manner by
covalently attaching one equivalent of a ligand, L
1, where FG
1 and FG
2 represent a functional group as defined above, and FG
2PG is a protected functional group to give an intermediate of formula (II). Deprotection
of the second functional group on the ligand, followed by reaction with a ligand L
2, which may be same or different than ligand L
1, then provides a compound of Formula (I). This method is suitable for preparing compounds
of Formula (I) where the ligands are the non-identical.
[0057] The ligands are covalently attached using conventional chemical techniques providing
for covalent linkage. Reaction chemistries resulting in such linkages are well known
in the art and involve the use of complementary functional groups as shown in Table
I below.
Table I
Representative Complementary Binding Chemistries |
First Reactive Group |
Second Reactive Group |
Linkage |
carboxyl |
amine |
amide |
sulfonyl halide |
amine |
sulfonamide |
hydroxyl |
alkyl/aryl halide |
ether |
hydroxyl |
isocyanate |
urethane |
amine |
epoxide |
β-hydroxyamine |
amine |
alkyl/aryl halide |
alkylamine |
amine |
isocyanate |
urea |
hydroxyl |
carboxyl |
ester |
amine |
aldehyde |
amine |
[0058] Reaction between a carboxylic acid and a primary or secondary amine in the presence
of suitable, well-known activating agents such as dicyclohexylcarbodiimide, results
in formation of an amide bond; reaction between an amine group and a sulfonyl halide,
in the presence of a base such as triethylamine, pyridine, and the like results in
formation of a sulfonamide bond; and reaction between an alcohol or phenol group and
an alkyl or aryl halide in the presence of a base such as triethylamine, pyridine,
and the like, results in formation of an ether bond.
[0059] A bivalent multibinding compound of Formula (II) where Ar
3 is the same as Ar
1, X is a bond, and Q is 2-hydroxyethylamino group, can be prepared from an acetophenone
derivative of formula
1 as shown in Scheme B below.
[0060] Condensation of an acetophenone derivative of formula
1 with a diamine of formula
2 in an ethereal solution such as tetrahydrofuran provides an imine of formula
3. Reduction of the imine with a suitable reducing agent such as borane provides a
compound of Formula (II). Suitable reaction solvents are tetrahydrofuran, and the
like. Compound
1 where Ar
1 is phenyl is prepared by heating acetophenone in 48% hydrobromic acid in dimethylsulfoxide.
[0061] Compounds of formula
1 can be prepared by methods well known in the art. For example, α,α-dihydroxy- 4-hydroxy-3-methoxycarbonylacetophenone
can be prepared by heating 5-acetylsalicylic acid methyl ester in 48% hydrobromic
acid.
[0062] Alternatively, a bivalent multibinding compound of Formula, (II) where Ar
3 is the same as Ar
1, X is a bond, and Q is 2-hydroxyethylamino group, can be prepared from an acetophenone
derivative of formula
1 as shown in Scheme C below.
[0063] A compound of Formula (II) can be prepared by reacting an epoxide of formula
4 with a diamine of formula
2. Epoxides
4 are either commercially available or they can be prepared by the methods described
in Kierstead, R.W. et. Al.
J. Med. Chem. 26, 1561-1569, (1983) or Hett, R. et. Al.
Tet. Lett.35, 9345-9348 (1994).
[0064] Another method of preparing a bivalent multibinding compound of Formula (II) where
the second ligand Ar
3 is the same as Ar
1, X is a bond, and Q is 2-hydroxyethylamino group, uses an (acetophenone derivative
of formula
5 as shown in Scheme D below.
[0065] Bromination of an acetophenone derivative of formula
5 with bromine in a halogenated organic solvent such as chloroform provides an α-bromoacetophenone
derivative of formula
6. Treatment of
6 with sodium azide followed by reduction of the resulting azide
7 with a suitable reducing agent such as lithium aluminum hydride provides ethanolamine
derivative of formula
8. Condensation of
2 equivalents of
8 with a dialdehyde compound
9 provides an imine of formula
10 which is converted to a compound of Formula (II) as described in Scheme A above.
Utility, Testing, and Administration
Utility
[0066] The multibinding compounds of this invention are β2 adrenergic receptor agonists.
Accordingly, the multibinding compounds and pharmaceutical compositions of this invention
are useful in the treatment and prevention of diseases mediated by β2 adrenergic receptor
such as asthma, bronchitis, and the like. They are also useful in the treatment of
nervous system injury and premature labor. It is also contemplated that the compounds
of this invention are useful for treating metabolic disorders such as obesity, diabetes,
and the like.
Testing
[0067] The β2 adrenergic receptor agonistic activity of the compounds of formula (I) to
may be demonstrated by a variety of
in vitro assays known to those of ordinary skill in the art, such as the assay described in
the biological examples 1 and 2. It may also be assayed by the Ex vivo assays described
in Ball, D. I. et al., "Salmterol a Novel, Long-acting beta 2-Adrenergic Agonist:
Characterization of Pharmacological Activity
in Vitro and
in Vivo"
Br. J. Pharmacol., 104, 665-671 (1991); Linden, A. et al., "Sameterol, Formaterol, and Salbutamol
in the Isolated Guinea-Pig Trachea: Differences in Maximum Relaxant Effect and Potency
but not in Functional Atagonism.
Thorax, 48, 547-553, (1993); and Bials, A. T. et al., Inventigations into Factors Determining
the Duration of Action of the Beta 2-Adrenoceptor Agonist, Salmateroal.
Br. J. Pharmacol., 108, 505-515 (1993); or in vivo assays such as those described in Ball, D. I. et
al., "Salmterol a Novel, Long-acting beta 2-Adrenergic Agonist: Characterization of
Pharmacological Activity
in Vitro and
in Vivo"
Br. J. Pharmacol., 104, 665-671 (1991); Kikkawa, H. et al., "RA-2005, a Novel, Long-acting, and Selective
Beta 2-Adrenoceptor Agonist: Characterization of its
in vivo Bronchodilating Action in Guinea Pigs and Cats in Comparison with other Beta 2-Agonists".
Biol. Pharm. Bull., 17, 1047-1052, (1994); and Anderson, G. P., "Formeterol: Pharmacology, Colecular
basis of Agonism and Mechanism of Long Duration of a Highly Potent and Selective Beta
2-Adrenoceptor Agonist Bronchodilator,
Life Sciences, 52, 2145-2160, (1993).
Pharmaceutical Formulations
[0068] When employed as pharmaceuticals, the compounds of this invention are usually administered
in the form of pharmaceutical compositions. These compounds can be administered by
a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous,
intramuscular, and intranasal. These compounds are effective as injectable inhaled
and oral compositions. Such compositions are prepared in a manner well known in the
pharmaceutical art and comprise at least one active compound.
[0069] This invention also includes pharmaceutical compositions which contain, as the active
ingredient, one or more of the compounds described herein associated with pharmaceutically
acceptable carriers. In making the compositions of this invention, the active ingredient
is usually mixed with an excipient, diluted by an excipient or enclosed within such
a carrier which can be in the form of a capsule, sachet, paper or other container.
When the excipient serves as a diluent, it can be a solid, semi-solid, or liquid material,
which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions
can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs,
suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium),
ointments containing, for example, up to 10% by weight of the active compound, soft
and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile
packaged powders.
[0070] In preparing a formulation, it may be necessary to mill the active compound to provide
the appropriate particle size prior to combining with the other ingredients. If the
active compound is substantially insoluble, it ordinarily is milled to a particle
size of less than 200 mesh. If the active compound is substantially water soluble,
the particle size is normally adjusted by milling to provide a substantially uniform
distribution in the formulation, e.g. about 40 mesh.
[0071] Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol,
mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin,
calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile
water, syrup, and methyl cellulose. The formulations can additionally include: lubricating
agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying
and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates;
sweetening agents; and flavoring agents. The compositions of the invention can be
formulated so as to provide quick, sustained or delayed release of the active ingredient
after administration to the patient by employing procedures known in the art.
[0072] The compositions are preferably formulated in a unit dosage form, each dosage containing
from about 0.001 to about 1 g, more usually about 1 to about 30 mg, of the active
ingredient. The term "unit dosage forms" refers to physically discrete units suitable
as unitary dosages for human subjects and other mammals, each unit containing a predetermined
quantity of active material calculated to produce the desired therapeutic effect,
in association with a suitable pharmaceutical excipient. Preferably, the compound
of Formula (I) above is employed at no more than about 20 weight percent of the pharmaceutical
composition, more preferably no more than about 15 weight percent, with the balance
being pharmaceutically inert carrier(s).
[0073] The active compound is effective over a wide dosage range and is generally administered
in a pharmaceutically effective amount. It, will be understood, however, that the
amount of the compound actually administered will be determined by a physician, in
the light of the relevant circumstances, including the condition to be treated, the
chosen route of administration, the actual compound administered and its relative
activity, the age, weight, and response of the individual patient, the severity of
the patient's symptoms, and the like.
[0074] For preparing solid compositions such as tablets, the principal active ingredient
is mixed with a pharmaceutical excipient to form a solid preformulation composition
containing a homogeneous mixture of a compound of the present invention. When referring
to these preformulation compositions as homogeneous, it is meant that the active ingredient
is dispersed evenly throughout the composition so that the composition may be readily
subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
This solid preformulation is then subdivided into unit dosage forms of the type described
above containing from, for example, 0.1 to about 500 mg of the active ingredient of
the present invention.
[0075] The tablets or pills of the present invention may be coated or otherwise compounded
to provide a dosage form affording the advantage of prolonged action. For example,
the tablet or pill can comprise an inner dosage and an outer dosage component, the
latter being in the form of an envelope over the former. The two components can be
separated by an enteric layer which serves to resist disintegration in the stomach
and permit the inner component to pass intact into the duodenum or to be delayed in
release. A variety of materials can be used for such enteric layers or coatings, such
materials including a number of polymeric acids and mixtures of polymeric acids with
such materials as shellac, cetyl alcohol, and cellulose acetate.
[0076] The liquid forms in which the novel compositions of the present invention may be
incorporated for administration orally or by injection include aqueous solutions,
suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with
edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil,
as well as elixirs and similar pharmaceutical vehicles.
[0077] Compositions for inhalation or insufflation include solutions and suspensions in
pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and
powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable
excipients as described
supra. Preferably the compositions are administered by the oral or nasal respiratory route
for local or systemic effect. Compositions in preferably pharmaceutically acceptable
solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled
directly from the nebulizing device or the nebulizing device may be attached to a
face mask tent, or intermittent positive pressure breathing machine. Solution, suspension,
or powder compositions may be administered, preferably orally or nasally, from devices
which deliver the formulation in an appropriate manner.
EXAMPLES
[0078] The following preparations and examples are given to enable those skilled in the
art to more clearly understand and to practice the present invention.
[0079] In the examples below, the following abbreviations have the following meanings. Unless
otherwise stated, all temperatures are in degrees Celsius. If an abbreviation is not
defined, it has its generally accepted meaning.
- Δ =
- Angstroms
- cm =
- centimeter
- DCC =
- dicyclohexyl carbodiimide
- DMF =
- N,N-dimethylformamide
- DMSO =
- dimethylsulfoxide
- g =
- gram
- HPLC =
- high performance liquid chromatography
- MEM =
- minimal essential medium
- mg =
- milligram
- MIC =
- minimum inhibitory concentration
- min =
- minute
- mL =
- milliliter
- mm =
- millimeter
- mmol =
- millimol
- N =
- normal
- THF =
- tetrahydrofuran
- µL =
- microliters
- Φm =
- microns
- rt =
- room temperature
- Rf =
- retention faction
- NMR =
- nuclear magnetic resonance
- ESMS =
- electrospray mass spectrum
- ppm =
- parts per million
Synthetic Examples
Example 1
Synthesis of trans-1,4-bis{N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}cyclohexane (following
figure 1)
[0080]
Step I
[0081] To a solution of 5-acetylsalicylic acid methyl ester
11 (5.0g, 25.7 mmole) in dimethylsulfoxide (44 mL) was added 48% hydrobromic acid. The
resulting mixture was stirred at 55 °C for 24 h, and poured into a slurry of ice-water
(~200 mL), precipitating a pale yellow solid. The solid was filtered, washed with
water (200 mL), and dried to give α,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12. The product was re-suspended in ethyl ether (~200 mL), filtered and dried to give
(3.41 g, 59%) of pure product. R
f= 0.8 (10% MeOH/CH
2Cl
2).
[0082] H
1-NMR (4/1 CDCl
3/CD
3OD, 299.96 MHz): δ (ppm) 8.73-8.72 (d, 1H), 8.28-8.24 (dd, 1H), 7.08-7.05 (d, 1H),
5.82 (s, 1H), 4.01 (s, 3H).
Step 2
[0083] To a suspension of α,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12 (0.3 g, 1.33 mmole) in THF (10 mL) was added a solution of
trans-1,4-diaminocyclohexane (76 mg, 0.66 mmole) in THF (5 mL). The resulting suspension
was stirred for 3 h at ambient temperature under nitrogen atmosphere, at which formation
of an imine was completed judged by TLC analysis. After cooling of the resulting solution
at ice bath, an excess amount of 2M BH
3-Me
2S in hexane (4 mL, 8 mmole) was added to the previous solution. The resulting mixture
was slowly warmed to rt and refluxed for 4 h under N
2 stream. After cooling the reaction mixture, MeOH (5 mL) was added to quench excess
amount of 2M BH
3-Me
2S. After stirring for 30 min., the final solution (or cloudy solution) was evaporated
in vacuo, yielding a pale brown solid. The solid was washed with EtOAc/hexane (1/2; 20 mL),
and dried. The crude product was dissolved in 50% MeCN/H
2O containing 0.5% TFA, and purified by prep-scale high performance liquid chromatography
(HPLC) using a linear gradient (5% to 50% MeCN/H
2O over 50 min, 20 mL/min; detection at 254 nM). Fractions with UV absorption were
analyzed by LC-MS to isolate
trans-1,4-bis{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}cyclohexane
13.
[0084] H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.35 (d, 2H), 7.18 (dd, 2H), 6.80-6.78 (d, 2H), 4.88-4.86
(m, 2H), 4.65 (s, 4H), 3.15 (br s, 4H), 2.89 (m, 2H), 1.68-1.55 (br m, 4H); ESMS (C
24H
34N
2O
6): calcd. 446.5, obsd. 447.5 [M+H]
+.
Compound 16:
[0085] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 1,8-diamino-
p-menthane gave 1,8-bis{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}-
p-menthane. ESMS (C
28H
42N
2O
6): calcd. 502.6, obsd. 503.3 [M+H]
+.
Compound 20:
[0086] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave 1-{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}-2-{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]benzene. ESMS (C
25H
30N
2O
6): calcd. 454.5, obsd 455.3 [M+H]
+.
Compound 21:
[0087] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-(4-aminophenyl)ethylamine gave 1-{2-[
N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-2-{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]benzene. ESMS (C
26H
32N
2O
6): calcd. 468.5, obsd. 469.3 [M+H]
+.
Compound 23:
[0088] Proceeding as described above but substituting
trans-1,4-diamino-cyclohexane with 2-aminobenzylamine gave 1-{
N-[2-{4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminomethyl}-4-{
N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino}benzene. ESMS (C
25H
30N
2O
6): calcd. 454.5, obsd. 455.5 [M+H]
+, 477.3 [M+Na]
+.
Example 2
Synthesis of 1-{2-[N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-{N-[2-phenyl-2-hydroxyethyl]amino]benzene (following figure 2)
[0089]
[0090] To a suspension of α,α-dihydroxy-4-hydroxy-3-methoxycarbonyl-acetophenone
12, prepared in Example 1, Step 1 above, (0.3 g, 1.33 mmole) in THF (10 mL) was added
a solution of 2-(4-aminophenyl)ethylamine
25 (0.181 g, 1.33 mmol) in THF (5 mL). The resulting suspension was stirred for 3 h
at ambient temperature under nitrogen atmosphere, followed by addition α,α-dihydroxy-acetophenone
24 (0.2g, 1.32 mmole). The reaction mixture was stirred for 3 h at RT, at which formation
of the imine was completed as judged by TLC analysis. The reaction mixture was cooled
in an ice bath and an excess amount of 2M BH
3-Me
2S in hexane (9 mL; 18 mmole) was added. The resulting mixture was slowly warmed to
rt, and refluxed for 4 h under N
2 stream. After cooling, MeOH (10 mL) was added to quench excess amount of BH
3-Me
2S. After stirring 30 min., at rt, the final solution (or cloudy suspension) was evaporated
in vacuo, to give a pale brown solid. The solid was washed with EtOAc/hexane (1/2; 20 mL),
and dried. The crude product was dissolved in 50% MeCN/H
2O containing 0.5% TFA, and purified by prep-scale high performance liquid chromatography
(HPLC) using a linear gradient (5% to 50% MeCN/H
2O over 50 min, 20 mL/min; detection at 254 nM). Fractions with UV absorption were
analyzed by LC-MS to locate 1-{2-[
N-2-[(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]-ethyl}-4-{
N-[2-phenyl-2-hydroxyethyl]amino]benzene
26. ESMS (C
25H
30N
2O
4): calcd. 422.5, obsd. 423.3 [M+H]
+.
Compound 27:
[0091] Proceeding as described above, but substituting α,α-dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone
with α,α-dihydroxyacetophenone gave 1-{2-[
N-[2-phenyl-2-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]-benzene.
ESMS (C
24H
28N
2O
8): calcd. 376.5, obsd. 377.0 [M+H]
+.
Example 3
Synthesis of 1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)amino]benzene (following figure 3)
[0092]
Step 1
[0093] To a solution of 4-(2-aminoethyl)aniline
25 (20 g, 147 mmole) in methanol (250 mL) was added (Boc)
2O (32.4 g, 148 mmole) in methanol (50 mL) at rt. After stirring for 24 h, the reaction
mixture was concentrated to dryness to afford a pale yellow oily residue. The oily
material solidified slowly; thus it was dissolved in 5% MeOH/CH
2Cl
2, and subsequently applied to flash silica column chromatography (3 to 10% MeOH/CH
2Cl
2). After purification, 4-(
N-Boc-2-aminoethyl)aniline
28 was obtained as a pale yellow solid (32.95g, 95%): R
f= 0.6 in 10% MeOH/CH
2Cl
2.
1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 6.96-6.93 (d, 2H), 6.69-6.65 (d, 2H), 3.20-3.13 (q, 2H),
2.63-2.58 (t, 2H), 1.41 (s, 9H).
Step 2
[0094] 4-(
N-Boc-2-aminoethyl)aniline
28 (1.25 g, 5.29 mmole) was dissolved in methanol (30 mL), followed by addition of phenyl
glyoxal
24 (0.708 g, 5.28 mmole). The reaction mixture was stirred for 1 h at rt, prior to addition
of NaCNBH
3 (0.665 g, 10.6 mmole). The final mixture was stirred for 12 h at rt, concentrated,
and purified by flash silica column chromatography (2 to 5% MeOH/CH
2Cl
2) to give
N-(2-phenyl-2-hydroxyethyl)-4-(
N-Boc-2-aminoethyl)-aniline as a pale yellow oil (1.71 g, 91%): R
f=0.18 in 5% MeOH/CH
2Cl
2.
1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.4-7.25 (m, 5H), 7.0-6.95 (d, 2H), 6.63-6.60 (d, 2H), 4.85-4.79
(dd, 1H), 3.3-3.21 (t, 2H), 3.2-3.15 (m, 2H), 2.64-2.5 (t, 2H), 1.42 (s, 9H).
Step 3
[0095] A solution of
N-(2-phenyl-2-hydroxyethyl)-4-(
N-Boc-2-aminoethyl)aniline (1.7 g, 4.77 mmole) in methylene chloride (10 mL) was cooled
in ice bath, and TFA (10 mL) was slowly added under a stream of nitrogen gas. The
reaction mixture was stirred for 1 h, and concentrated to yield a pale yellow oil.
The crude material was purified by reversed phase HPLC (10% to 40% MeCN/H
2O over 50 min; 20 mL/min) to give
N-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline 29 as the TFA salt (1.1 g).
1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.42-7.3 (m, 5H), 7.29-7.25 (d, 2H), 7.12-7.0 (d, 2H), 4.85-4.82
(m, 1H), 3.45-3.35 (m, 2H), 3.18-3.1 (t, 2H), 2.98-2.94 (t, 2H); ESMS (C
16H
20N
2O
1): calcd. 256.4, obsd. 257.1 [M+H]
+, 278.8 [M+Na]
+, 513.4 [2M+H]
+.
Step 4
[0096] To a solution of
N-(2-phenyl-2-hydroxyethyl)-4-(2-aminoethyl)aniline trifluoroacetate salt
29 (1.1 g, 2.3 mmole) in methanol (10 mL) was added 5 M NaOH solution (0.93 mL). After
stirring for 10 min., the solution was concentrated to dryness. The residue was dissolved
in THF (25 mL), and α,α-dihydroxy-4-hydroxy-3-methoxy-carbonylacetophenone
12 (0.514 g, 2.27 mmole) was added. The reaction mixture was stirred for 12 h at rt,
cooled to 0 °C, and BH
3/Me
2S (1.14 mL, 10 M) was added under nitrogen atmosphere. The reaction mixture was gradually
warmed to rt, stirred for 2 h at rt, and refluxed for 4 h. The reaction mixture was
cooled and methanol (10 mL) was added slowly. After stirring for 30 min., at rt, the
reaction mixture was concentrated to afford a solid residue, which was dissolved in
MeOH (20 mL) containing 10% TFA. Evaporation of the organics yielded a pale yellow
oil which was purified by reversed phase HPLC: 10% to 30% MeCN/H
2O over 50 min; 20 mL/min to give 1-{2-[N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]-amino]ethyl}-4-[N-(2-phenyl-2-hydroxyethyl)-amino]benzene
30 as the TFA salt (0.65 g).
1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.42-7.3 (m, 6H), 7.28-7.24 (d, 2H), 7.18-7.14 (dd, 1H),
7.1-7.07 (d, 2H), 6.80-6.77 (d, 1H), 4.86-4.82 (m, 2H), 4.65 (s, 2H), 3.44-3.34 (m,
2H), 3.28-3.22 (m, 2H), 3.20-3.14 (m, 2H), 3.04-2.96 (m, 2H); ESMS (C
25H
30N
2O
4): calcd. 422.5, obsd. 423.1 [M+H]
+, 404.7 [M-1H
2O]
+, 387.1 [M-2H
2O]
+.
Example 4
Synthesis of 1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]benzene (following figure 4)
[0097]
Step 1
[0098] A solution of 4-(
N-Boc-2-aminoethyl)aniline
28 (7.0 g, 29.6 mmole) in ethanol (100 mL) and (
R)-styreneoxide (3.56 g, 29.6 mmole) was refluxed for 24 h. The organics were removed
to give a pale yellow solid.
N-(2-phenyl-2-(
S)-hydroxyethyl)-4-(
N-Boc-2-aminoethyl)aniline was separated by flash silica column chromatography: 1/2
EtOAc/hexane to 3/1 EtOAc/hexane to 3% MeOH in 3/1 EtOAc/hexane: Rf = 0.39 in 3% MeOH/CH
2Cl
2.
Step 2
[0099] A solution of
N-(2-phenyl-2-(
S)-hydroxyethyl)-4-(
N-Boc-2-aminoethyl)-aniline (2.5 g, 7.0 mmole) in CH
2Cl
2 (15 mL) was cooled in an ice bath under stream of nitrogen and TFA (15 mL) was slowly
added. The reaction mixture was stirred for 2 h at 0°C and then concentrated
in vacuo. The crude product was dissolved in 20% MeCN/H
2O and purified by preparative reversed phase HPLC (5 to 2% MeCN/H
2O over 50 min; 254 nm; 20 mL/min.), to give
N-(2-phenyl-2-(
S)-hydroxyethyl)-4-(2-aminoethyl)aniline trifluoroacetate salt
31 as a colorless oil.
1H-NMR (CD
3OD, 299.96 MHz): δ (ppm); 7.45-7.25 (m, 9H), 4.9 (dd, 1H), 3.55-3.45 (m, 2H), 3.21-3.15
(t, 2H), 3.05-2.95 (t, 2H) ESMS (C
16H
20N
2O
1): calcd. 256.4, obsd. 257.1 [M+H]
+, 280.2 [M+Na]
+.
Step 3
[0100] To a solution of
N-(2-phenyl-2-(
S)-hydroxyethyl)-4-(2-aminoethyl)aniline trifluoroacetate
31 (0.144 g, 0.3 mmole) in methanol (10 mL) was added aq. NaOH solution (1.0 M, 0.625
mL). The solution was concentrated to dryness and the residue was dissolved in anhydrous
THF (5 mL). α,α-Dihydroxy-4-hydroxy-3-methoxycarbonylacetophenone
12 (0.067 g, 0.3 mmole) was added and the reaction mixture was stirred for 12 h at rt.
BH
3-Me
2S (0.2 mL, 2M) was added at 0°C and the reaction mixture was heated at 75 °C for 6
h. After cooling the reaction mixture in ice bath, MeOH (5 mL) was slowly added to
it to quench the reaction, and the reaction mixture was stirred for 30 min., at rt.
The organics were removed and the residue was dissolved in TFA/MeOH (1/9; 20 mL),
and concentrated. The crude product was dissolved in 20% MeCN/H
2O, and purified by preparative HPLC: 5 to 20% MeCN/H
2O; 20 mL/min; 254 nm.) to give 1-{2-[
N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)-amino]benzene 33.
[0101] 1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.42-7.29 (m, 8H), 7.22-7.18 (d, 2H), 7.17-7.14 (dd, 1H),
6.80-6.77 (d, 1H), 4.9-4.85 (m, 2H), 4.65 (s, 2H), 3.5-3.34 (m, 2H), 3.28-3.25 (m,
2H), 3.19-3.14 (m, 2H), 3.04-2.98 (m, 2H); ESMS (C
25H
30N
2O
4): calcd. 422.5, obsd. 423.1 [M+H]
+, 446.1 [M+Na]
+.
[0102] Proceeding as described in Example 4 above but substituting (
R)-styreneoxide with (
S)-styreneoxide gave 1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-phenyl-2-(
R)-hydroxyethyl)amino]benzene
34.
[0103] 1H-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.42-7.28 (m, 8H), 7.20-7.1 (m, 3H), 6.80-6.77 (d, 1H), 4.9-4.85
(m, 2H), 4.65 (s, 2H), 3.45-3.34 (m, 2H), 3.28-3.25 (m, 2H), 3.19-3.15 (m, 2H), 3.04-2.98
(m, 2H); ESMS (C
25H
30N
2O
4): calcd. 422.5, obsd. 423.1 [M+H]
+, 446.1 [M+Na]
+.
Example 5
Synthesis of 1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(R)-hydroxyethyl]aminoethyl}-4-[N-(2-phenyl-2-(S)-hydroxyethyl)amino]phenyl (following figure 5)
[0104]
Step 1
[0105] A mixture of 4-(
N-Boc-2-aminoethyl)aniline
28 (10 g, 42.34 mmole), benzaldehyde (4.52 mL, 44.47 mmole), and molecular sieves 4A
(10 g) in toluene (100 mL) was refluxed at 95 °C for 15 h. The reaction mixture was
filtered and the filtrate was concentrated
in vacuo to give a colorless oil. The oil was dissolved in MeOH (150 mL) and AcOH (0.5 mL)
and NaCNBH
3 (2.79 g, 44.4 mmole) were added. The reaction mixture was stirred at 0°C for 1 h
and at rt for 2 h and then concentrated
in vacuo to give a pale yellow oily residue. Purification by flash silica column chromatography:
1/1 hexane/EtOAc gave
N-benzyl-4-(
N-Boc-2-aminoethyl)aniline
41 as colorless oil (11.5 g, 83%). R
f=0.75 in 1/1 hexane/EtOAc. H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.38-7.2 (m, 5H), 6.87-6.84 (d, 2H), 6.58-6.55 (d, 2H), 4.27
(s, 2H), 3.2-3.15 (m, 2H), 2.6-2.56 (t, 2H), 1.41 (s, 9H); ESMS (C
20H
26N
2O
2): calcd. 326.4, obsd. 328 [M+H]
+.
Step 2
[0106] A mixture of
N-benzyl-4-(
N-Boc-2-aminoethyl)aniline
41 (10 g, 30.7 mmole) and (
R)-styreneoxide (3.51 mL, 30.7 mmole) in EtOH (100 mL) was refluxed for 48 h. A small
aliquot of the reaction mixture was taken out for liquid chromatographic analysis,
which indicated that the desired adduct 2-[(
N-benzyl-4-[2-
N-Boc-aminoethyl)anilino]-1-phenylethanol was formed as a minor product along with
another regio-isomer 2-[(
N-benzyl-4-[2-
N-Boc-aminoethyl)anilino]-2-phenyl-ethanol in a ratio of ~1/2. Evaporation of the solution
afforded thick, pale yellow oil, which was purified by flash silica column chromatography:
4/1 to 2/1 hexane/EtOAc. After repeated chromatography, 2-[(
N-benzyl-4-[2-
N-Boc-aminoethyl)anilino]-1-phenyl-ethanol was obtained as a colorless oil (4.01 g,
29%) (R
f= 0.76 in 2/1 hexane/EtOAc). H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.4-7.1 (m, 10H), 7.1-7.06 (d, 2H), 6.68-6.65 (d, 2H), 5.0
(t, 1H), 4.52-4.46 (d, 1H), 4.26-4.22 (d, 1H), 3.76-3.68 (dd, 1H), 3.56-3.48 (dd,
1H), 3.22-3.12 (m, 2H), 2.68-2.56 (m, 2H), 1.41 (s, 9H); ESMS (C
28H
34N
2O
3): calcd. 446.6, obsd. 447.1 [M+H]
+, 893.4 [2M+H]
+.
Step 3
[0107] To a solution of 2-[(
N-benzyl-4-[2-
N-Boc-aminoethyl)anilino]-1-phenyl-ethanol (4.01 g, 8.99 mmole) in CH
2Cl
2 (15 mL) maintained in an ice bath was added TFA (15 mL) under stream of nitrogen
atmosphere. After stirring at 0 °C for 30 min., the reaction mixture was concentrated
in vacuo, yielding a pale yellow oil. Purification by flash silica column chromatography:
(½ hexane/EtOAc to 5%
i-PrNH
2 in ½ hexane/EtOAc) gave 2-[(
N-benzyl-4-[2-aminoethyl)anilino]-1-phenyl-ethanol
42 as a pale yellow oil from such fractions with R
f of 0.2 (5%
i-PrNH
2 in ½ hexane/EtOAc) in 74% yield (2.29 g). H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.38-7.06 (m, 10H), 7.01-6.98 (d, 2H), 6.71-6.68 (d, 2H),
5.02-4.96 (dd, 1H), 4.54-4.48 (d, 1H), 4.29-4.23 (d, 1H), 3.76-3.67 (dd, 1H), 3.58-3.50
(dd, 1H), 2.82-2.74 (t, 2H), 2.64-2.59 (t, 2H); ESMS (C
23H
26N
2O
1): calcd. 346.5, obsd. 346.3[M]
+,
Step 4
[0108] A mixture of 2-[(
N-benzyl-4-[2-aminoethyl)anilino]-1-phenylethanol
42 (2.28 g, 6.59 mmole), benzaldehyde (0.74 mL, 7.28 mmole), and molecular sieves 4A
(4 g) in toluene (40 mL) was heated at 90 °C for 14 h. The reaction mixture was cooled
and filtered, and the sieves were rinsed with toluene. The combined filtrates were
concentrated to give an oily residue which was washed with hexane, and dried. The
residue was dissolved in MeOH (40 mL) containing AcOH (0.4 mL) and the reaction mixture
was cooled in an ice bath. NaCNBH
3 (0.62 g, 9.87 mmole) was added and the reaction mixture was stirred for 2 h at rt,
and then concentrated. The oily residue was dissolved in 60% MeCN/H
2O, and purified by reversed phase preparative liquid chromatography (40 to 80% MeCN/H
2O over 30 min; 30 mL/min) to give 2-[(
N-benzyl-4-[2-
N-benzylaminoethyl)anilino]-1-phenylethanol as the TFA salt. The product was treated
with alkaline brine solution, and extracted with ether (200 mL). The organic layer
was dried with NaSO
4, and concentrated, to give 2-[(
N-benzyl-4-[2-
N-benzylaminoethyl)anilino]-1-phenylethanol
43 as a colorless oil (1.36 g). H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.36-7.06 (m, 15H), 6.98-6.95 (d, 2H), 6.69-6.60 (d, 2H),
5.01-4.96 (t, 1H), 4.54-4.47 (d, 1H), 4.29-4.24 (d, 1H), 3.73 (s, 2H), 3.72-3.68 (dd,
1H), 3.59-3.54 (dd, 1H), 2.80-2.74 (m, 2H), 2.70-2.64 (m, 2H); ESMS (C
30H
32N
2O
1): calcd. 436.6, obsd. 437.2 [M+H]
+.
Step 5
[0109] A concentrated solution of 2-[(
N-benzyl-4-[2-
N-benzylaminoethyl)anilino]-1-phenylethanol (1.36 g, 3.12 mmole) and compound (
S)-4-benzyloxy-3-methoxycarbonylstyreneoxide
44 (0.887 g, 3.12 mmole; ~95% ee) (prepared as described in R. Hett, R. Stare, P. Helquist,
Tet. Lett.,
35, 9375-9378, (1994)) in toluene (1 mL) was heated at 105 °C for 72 h under nitrogen
atmosphere. The reaction mixture was purified by flash silica column chromatography
(2/1 hexane/EtOAc to 3% MeOH in 1/1 hexane/EtOAc) to give 1-{2-[
N-benzyl-
N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(
R)-hydroxy]ethylaminoethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxy)ethylamino]benzene
45. (R
f= 0.62 in 3% MeOH in 1/1 hexane/EtOAc) was obtained as a pale yellow foam (2.0 g,
89%).
[0110] H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.67-7.66 (d, 1H), 7.49-7.42 (m, 2H), 7.38-7.0 (m, 20H),
6.88-6.85 (d, 2% 6.65-6.62 (d, 2H), 5.15 (s, 2H), 5.05-4.98 (t, 1H), 4.6-4.54 (t,
1H), 4.53-4.46 (d, 1H), 4.28-4.22 (d, 1H), 3.84 (s, 3H), 3.72-3.64 (m, 3H), 3.56-3.46
(dd, 1H), 2.74-2.56 (m, 6H); ESMS (C
47H
48N
2O
5): calcd. 720.9, obsd. 721.4 [M+H]
+, 743.3 [M+Na]
+.
Step 6
[0111] To a suspension of LiAlH
4 (0.21 g, 5.56 mmole) in THF (40 mL) cooled with ice bath was added 1-{2-[
N-benzyl-
N-2-(4-benzyloxy-3-methoxycarbonylphenyl)-2-(
R)-hydroxyethyl]aminoethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)amino]benzene
45 (2.0 g, 2.78 mmole) in THF (10 mL) under nitrogen atmosphere. The reaction mixture
was warmed slowly to rt and the stirring was continued for 5 h. The reaction was cooled
to 0°C, and 10% NaOH (0.5 mL) was slowly added. After 30 min., a thick gel formed.
The gel was diluted with THF (300 mL), filtered, and the solid mass was rinsed with
THF (50 mL). The filtrates were combined, and concentrated
in vacuo, yielding an oily residue.
[0112] The residue was purified by flash silica column chromatography (2/1 hexane/EtOAc
to 3% MeOH in 1/1 hexane/EtOAc) to give 1-{2-[
N-benzyl-
N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(
R)-hydroxyethyl]aminoethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)amino]benzene as a colorless oil (1.28 g, 67%). H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.4-7.0 (m, 22H), 6.85-6.82 (m, 3H), 6.63-6.60 (d, 2H), 5.02-4.94
(m, 3H), 4.66 (s, 2H), 4.59-4.54 (dd, 1H), 4.48-4.4 (d, 1H), 4.24-4.16 (d, 1H), 3.76-3.7
(d, 1H), 3.69-3.62 (dd, 1H), 3.58-3.52 (d, 1H), 3.50-3.44 (dd, 1H), 2.76-2.54 (m,
6H); ESMS (C
46H
48N
2O
4): calcd. 692.90, obsd. 693.5 [M+H]
+.
Step 7
[0113] A solution of 1-{2-[
N-benzyl-
N-2-(4-benzyloxy-3-hydroxymethylphenyl)-2-(
R)-hydroxyethyl]amino]ethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)amino]-benzene (1.28 g, 1.85 mmole) in EtOH (80 mL) was hydrogenated
under H
2 (1 atm) with 10% Pd/C (0.6 g) for 36 h. After filtration and rinsing of the catalyst
with EtOH (50 mL), the filtrates were combined, and evaporated
in vacuo, yielding pale yellow foam which was dissolved in 10% MeCN/H
2O, and purified by reversed phase preparative liquid chromatography (10 to 30% MeCN/H
2O (containing 0.3% TFA) over 50 min; 30 mL/min; 254 nm) to give 1-{2-[
N-2-(4-hydroxy-3-hydroxymethyl-phenyl)-2-(
R)-hydroxyethyl]aminoethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)-amino]benzene as the TFA salt (0.6 g, 50%). Optical purity of 1-{2-[
N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-(
R)-hydroxyethyl]aminoethyl}-4-[
N-(2-phenyl-2-(
S)-hydroxyethyl)amino]benzene
46 which was analyzed with capillary electrophoresis by using a chiral medium, and estimated
to be ~93%.
[0114] H
1-NMR (CD
3OD, 299.96 MHz): δ (ppm) 7.42-7.28 (m, 8H), 7.26-7.22 (d, 2H), 7.18-7.14 (dd, 1H),
6.80-6.77 (d, 1H), 4.88-4.82 (m, 2H), 4.65 (s, 2H), 3.5-3.43 (m, 2H), 3.29-3.26 (m,
2H), 3.19-4.14 (m, 2H), 3.06-3.0 (m, 2H); ESMS (C
25H
30N
2O
4): calcd. 422.5, obsd. 423.1 [M+H]
+, 445.4 [M+Na]
+,
Example 6
Synthesis of 1-{6-[N-[2-(4-hydroxy-3-hydroxymethylphenyl)-2-[hydroxyethyl]-amino]hexyloxy}-4-{6-[N-[2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]hexyloxypropyl}benzene
(following figure 6)
[0115]
Step 1
[0116] A solution of 3-(4-hydroxyphenyl)-1-propanol (2.0 g, 13.1 mmole) in DMF (5 mL) was
added to a solution of DMF (35 mL) containing NaH (1.31 g, 60% in mineral oil) at
0°C under nitrogen atmosphere. The reaction mixture was slowly warmed to 80 °C. After
stirring for 1 h at 80 °C, the reaction mixture was cooled to 0 °C, and 6-bromohexanenitrile
(5.78 g, 32.83 mmole) was added. The final mixture was re-heated to 80 °C, and stirred
for 24 h. The reaction mixture was quenched with saturated NaCl solution (200 mL),
and the product was extracted with EtOAc (300 mL). The organic layer was washed with
brine solution, dried with Na
2SO
4, and evaporated to dryness, yielding a pale yellow solid. Purification of the crude
product by flash silica column chromatography: 4/1 to 1/1 hexane/EtOAc provided 6-{3-[4-(5-cyanopentyloxy)phenyl]propoxy}hexanenitrile
in 30% yield (1.33 g). R
f= 0.63 in 1/1 EtOAc/hexane.
1H-NMR (CDCl
3, 299.96 MHz): δ (ppm) 7.09-7.07 (d, 2H), 6.81-6.78 (d, 2H), 3.96-3.92 (t, 2H), 3.42-3.37
(m, 4H), 2.64-2.58 (t, 2H), 2.39-2.32 (m, 4H), 1.87-1.52 (m, 14 H).
Step 2
[0117] A solution of 6-{3-[4-(5-pentyloxy)phenyl]propoxy}hexanenitrile (1.33 g, 3.88 mmole)
in THF (10 mL) was added to a solution of LiAlH
4 (0.442 g, 11.65 mmole) in THF (50 mL) at 0 °C under nitrogen atmosphere. The reaction
mixture was heated slowly to reflux, and stirred for 2 h. The reaction mixture was
cooled to 0 °C, and 10% NaOH solution (5 mL) was slowly added. After 30 min., the
reaction mixture was filtered, and the collected solids were washed with THF (100
mL). The filtrate was concentrated to yield a pale yellow oil which was purified by
flash silica column chromatography: 5% MeOH/CH
2Cl
2 to 3%
i-PrNH
2/20% MeOH/CH
2Cl
2 to give 6-{3-[4-(6-aminohexyloxy)-phenyl]propoxy}-hexylamine as a colorless oil (0.5
g, 37%) which was converted to the desired compound by proceeding as described in
Example 1, step 2 above. The crude product was purified by preparatory reversed phase
HPLC: 10 to 40% MeCN/H
2O over 40 min; 20 mL/min; 254 nm. ESMS (C
39H
58N
2O
8): calcd. 682.8, obsd. 683.6 [M+H]
+, 797.5 [M+CF
3CO
2H]
+.
Example 7
Synthesis of 1-{2-[N-2-(4-hydroxy-3-hydroxymethylphenyl)-2-hydroxyethyl]amino]ethyl}-4-[N-(2-napth-1-yloxymethyl-2-hydroxyethyl)amino]benzene (following figure 7)
[0118]
Step 1
[0119] A solution of EtOH (50 mL) containing 4-(
N-Boc-2-aminoethyl)aniline
28 (0.4 g, 1.69 mmole) and 3-(1-naphthoxy)-1,2-epoxypropane
55 (0.33 g, 1.65 mmole) was refluxed for 18 h, and concentrated
in vacuo to dryness, yielding a pale yellow oil. It was dissolved in 10 mL of CH
2Cl
2, cooled in ice bath, and treated with TFA (5 mL). After stirring for 2 h at 0°C,
the mixture was evaporated, yielding a pale red oil. It was dissolved in 30% aqueous
acetonitrile, and purified by preparatory HPLC: 10 to 30% MeCN/H
2O over 30 min; 20 mL/min; 254 nm. The product
56 was obtained as colorless oil (260 mg; TFA salt). H
1-NMR (CD
3OD, 299.96 MHz): (ppm) 8.88-8.25 (dd, 1H), 7.82-7.79 (dd, 1H), 7.51-7.42 (m, 3H),
7.39-7-38 (d, 1H), 7.33-7.30 (d, 2H), 7.25-7.23 (d, 2H), 6.91-6.89 (d, 1H), 4.37-4.31
(m, 1H), 4.22-4.19 (m, 2H), 3.69-3.63 (dd, 1H), 3.67-3.54 (dd, 1H), 3.17-3.11 (t,
2H), 2.96-2.91 (t, 2H); ESMS (C
21H
24N
2O
2): calcd. 336.4, obsd. 337.5 [M+H]
+, 359.6 [M+Na]
+, 673.4 [2M+H]
+.
Step 2
[0120] To a solution of compound
56 (0.13 g, 0.023 mmole; TFA salt) in 5 mL of MeOH was added 1.0 M NaOH (1.0 M, 0.46
mL). After homogeneous mixing, the solution was evaporated to dryness. The residue
was dissolved in THF (10 mL), followed by addition of glyoxal
12 (52 mg; 0.023 mmole). The resulting suspension was stirred for 4 h at ambient temperature
under nitrogen atmosphere. After cooling of the resulting solution in ice bath, an
excess amount of 2M BH
3-Me
2S in THF (3 mL; 6 mmole) was added to the previous reaction solution. The resulting
mixture was slowly warmed to rt, and refluxed for 4 h under N
2 stream. After cooling of the hot solution, 5 mL of MeOH was added to the cooled mixture
to quench the reaction mixture under nitrogen atmosphere. After stirring 30 min at
rt, the final solution was evaporated
in vacuo, yielding a pale brown solid. It was washed with EtOAc/hexane (1/2; 20 mL), and dried.
The crude product was dissolved in 50% MeCN/H
2O containing 0.5% TFA, and purified by prep-scale high performance liquid chromatography
(HPLC) using a linear gradient (5% to 50% MeCN/H
2O over 50 min, 20 mL/min; detection at 254 nM). Fractions with UV absorption were
analyzed by LC-MS to locate the desired product 1-{2-[
N-2-(4-hydroxy-3-hydroxy-methylphenyl)-2-hydroxyethyl]amino]-ethyl}-4-[
N-(2-napth-1-yloxymethyl-2-hydroxy-ethyl)amino]benzene
57. ESMS (C
30H
34N
2O
5): calcd. 502.6, obsd. 503.2 [M+H]
4, 525.6 [M+Na]
+.
Formulation Examples
Example 1
[0121] Hard gelatin capsules containing the following ingredients are prepared:
Ingredient |
Quantity (mg/capsule) |
Active Ingredient |
30.0 |
Starch |
305.0 |
Magnesium stearate |
5.0 |
[0122] The above ingredients are mixed and filled into hard gelatin capsules in 340 mg quantities.
Example 2
[0123] A tablet Formula is prepared using the ingredients below:
Ingredient |
Quantity (mg/tablet) |
Active Ingredient |
25.0 |
Cellulose, microcrystalline |
200.0 |
Colloidal silicon dioxide |
10.0 |
Stearic acid |
5.0 |
[0124] The components are blended and compressed to form tablets, each weighing 240 mg.
Example 3
[0125] A dry powder inhaler formulation is prepared containing the following components:
Ingredient |
Weight % |
Active Ingredient |
5 |
Lactose |
95 |
[0126] The active ingredient is mixed with the lactose and the mixture is added to a dry
powder inhaling appliance.
Example 4
[0127] Tablets, each containing 30 mg of active ingredient, are prepared as follows:
Ingredient |
Quantity (mg/tablet) |
Active Ingredient |
30.0 mg |
Starch |
45.0 mg |
Microcrystalline cellulose |
35.0 mg |
Polyvinylpyrrolidone (as 10% solution in sterile water) |
4.0 mg |
Sodium carboxymethyl starch |
4.5 mg |
Magnesium stearate |
0.5 mg |
Talc |
1.0 mg |
Total |
|
[0128] The active ingredient, starch and cellulose are passed through a No. 20 mesh U.S.
sieve and mixed thoroughly. The solution of polyvinylpyrrolidone is mixed with the
resultant powders, which are then passed through a 16 mesh U.S. sieve. The granules
so produced are dried at 50E to 60EC and passed through a 16 mesh U.S. sieve. The
sodium carboxymethyl starch, magnesium stearate, and talc, previously passed through
a No. 30 mesh U.S. sieve, are then added to the granules which, after mixing, are
compressed on a tablet machine to yield tablets each weighing 120 mg.
Example 5
[0129] Capsules, each containing 40 mg of medicament are made as follows:
Ingredient |
Quantity (mg/capsule) |
Active Ingredient |
40.0 mg |
Starch |
109.0 mg |
Magnesium stearate |
1.0 mg |
Total |
|
[0130] The active ingredient, starch, and magnesium stearate are blended, passed through
a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 150 mg quantities.
Example 6
[0131] Suppositories, each containing 25 mg of active ingredient are made as follows:
Ingredient |
Amount |
Active Ingredient |
25 mg |
Saturated fatty acid glycerides to |
2,000 mg |
[0132] The active ingredient is passed through a No. 60 mesh U.S. sieve and suspended in
the saturated fatty acid glycerides previously melted using the minimum heat necessary.
The mixture is then poured into a suppository mold of nominal 2.0 g capacity and allowed
to cool.
Example 7
[0133] Suspensions, each containing 50 mg of medicament per 5.0 mL dose are made as follows:
Ingredient |
Amount |
Active Ingredient |
50.0 mg |
Xanthan gum |
4.0 mg |
Sodium carboxymethyl cellulose (11%) |
|
Microcrystalline cellulose (89%) |
50.0 mg |
Sucrose |
1.75 g |
Sodium benzoate |
10.0 mg |
Flavor and Color |
q.v. |
Purified water to |
5.0 mL |
[0134] The active ingredient, sucrose and xanthan gum are blended, passed through a No.
10 mesh U.S. sieve, and then mixed with a previously made solution of the microcrystalline
cellulose and sodium carboxymethyl cellulose in water. The sodium benzoate, flavor,
and color are diluted with some of the water and added with stirring. Sufficient water
is then added to produce the required volume.
Example 8
[0135] A formulation may be prepared as follows:
Ingredient |
Quantity (mg/capsule) |
Active Ingredient |
15.0 mg |
Starch |
407.0 mg |
Magnesium stearate |
3.0 mg |
Total |
|
[0136] The active ingredient, starch, and magnesium stearate are blended, passed through
a No. 20 mesh U.S. sieve, and filled into hard gelatin capsules in 425.0 mg quantities.
Example 9
[0137] A formulation may be prepared as follows:
Ingredient |
Quantity |
Active Ingredient |
5.0 mg |
Corn Oil |
1.0 mL |
Example 10
[0138] A topical formulation may be prepared as follows:
Ingredient |
Quantity |
Active Ingredient |
1-10 g |
Emulsifying Wax |
30 g |
Liquid Paraffin |
20 g |
White Soft Paraffin |
to 100 g |
[0139] The white soft paraffin is heated until molten. The liquid paraffin and emulsifying
wax are incorporated and stirred until dissolved. The active ingredient is added and
stirring is continued until dispersed. The mixture is then cooled until solid.
[0140] Another preferred formulation employed in the methods of the present invention employs
transdermal delivery devices ("patches"). Such transdermal patches may be used to
provide continuous or discontinuous infusion of the compounds of the present invention
in controlled amounts. The construction and use of transdermal patches for the delivery
of pharmaceutical agents is well known in the art.
See, e.
g., U.S. Patent 5,023,252, issued June 11, 1991, herein incorporated by reference in
its entirety. Such patches may be constructed for continuous, pulsatile, or on demand
delivery of pharmaceutical agents.
[0141] Other suitable formulations for use in the present invention can be found in
Remington's Pharmaceutical Sciences, edited by E. W. Martin (Mack Publishing Company, 18th ed., 1990).
Biological Examples
Example I
β2-Adrenergic Receptor In Vitro Functional Assay
[0142] The β2-adrenergic receptor functional activity of compounds of the invention was
tested follows.
Cell Seeding and Growth:
[0143] Primary bronchial smooth muscle cells from a 21 yr. old male (Clonetics, San Diego,
CA) were seeded at 50,000 cells/well in 24-well tissue culture plates. The media used
was Clonetic's SmBM-2 supplemented with hEGF, Insulin, hFGF, and Fetal Bovine Serum.
Cells were grown two days at 37°C, 5% CO
2 until confluent monolayers were seen.
Agonist Stimulation of Cells
[0144] The media was aspirated from each well and replaced with 250 ml fresh media containing
1mM IBMX, a phospodiesterase inhibitor (Sigma, St Louis, MO). Cells were incubated
for 15 minutes at 37 °C, and then 250 ml of agonist at appropriate concentration was
added. Cells were then incubated for an additional 10 minutes. Media was aspirated
and 500 ml cold 70% EtOH was added to cells, and then removed to an empty 96-well
deep-well plate after about 5 minutes. This step was then repeated. The deep-well
plate was then spun in a speed-vac until all EtOH dried off, leaving dry pellets.
cAMP (pmol/well) was quantitated using a cAMP ELISA kit from Stratagene (La Jolla,
CA). EC
50 curves were generated using the 4-parameter fit equation:
where,
y = cpm a = total binding c = IC
50
x = [compound] d = NS binding b = slope
Fix NS binding and allow all other parameters to float.
Example 2
β2-Adrenergic Receptor In Vitro Radioligand Binding Assay
[0145] The β1/2-adrenergic receptor binding activity of compounds of the invention can be
tested follows. SF9 cell membranes containing either β1 or β2-adrenergic receptor
(NEN, Boston, MA) were incubated with 0.07 nM
125I-iodocyanopindolol (NEN, Boston, MA) in binding buffer containing 75mM Tris-HCl (pH
7.4), 12.5 mM MgCl
2 and 2 mM EDTA and varying concentrations of test compounds or buffer only (control)
in 96-well plates. The plates were incubated at room temperature with shaking for
1 hour. The receptor bound radioligand was harvested by filtration over 96-well GF/B
filter plates (Packard, Meriden, CT) pre-blocked with 0.3%polyethylenimine and washed
twice with 200Φ1 PBS using cell harvester. The filters were washed thrice with 200Φ1
PBS using cell harvester and then resuspended in 40Φ1 scintillation cocktail. The
filter-bound radioactivity was measured with a scintillation counter and IC
50 curves are generated using the standard 4-parameter fit equation described above.